One of the most pervasive problems in the use of recombinant retroviruses for gene therapy is maintaining transgene expression after cellular infection. In mitotically active cells in culture, such as skin fibroblasts, hepatocytes, or astroglia, retroviruses readily infect and express the integrated transgene. However, when these cells are allowed to reach a post-mitotic state (either in vitro or after transplantation in vivo), expression of the retroviral transcriptional cassette is blocked. Similarly, when retroviral genomes are introduced into embryonic cells, either by normal viral infection or by injection of proviral DNA into the pronucleus, the virus is able to integrate into the host genome, but transgenes introduced in this way are not expressed.
Cells with regulated transgenes have historically been produced by the simultaneous integration of several plasmids into the host cell by transfection. This results in the formation of very few stably transduced cells having all of the required components for regulation intact. Moreover, since variable numbers of plasmids integrate into the host cell genome, individual cells may have very different inductive properties.
To date, no retroviral systems have been described in the art in which transgene expression is regulated through the introduction of novel ligand-activated and/or constitutively active response elements into the LTR thereof. While ligand stimulated transcription systems have been described in plasmids and retroviruses using the tetracycline repressor, these vectors are designed with responsive internal promoters and, therefore, are presumably susceptible to the same LTR transcriptional blockage observed for other retroviral vectors.
Accordingly, what is needed in the art are viruses that have potentially much greater range of application to gene therapy, production of transgenic animals, and gene transfer to developing embryos than previously available retroviral vectors.